This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The capacity for precise tuning of active site reduction potentials in folded polypeptide environments has afforded nature the freedom to access myriad chemistries despite its limited repertoire of incorporated elements. The reduction potential of Pseudomonas aeruginosa azurin has long been known to be sensitive to substitutions within both the inner and outer type 1 copper coordination sphere. This sensitivity makes it possible to tune the Cu(II/I) potential over 0.5 V higher than the aqueous redox couple. In order to further explore this control over the Cu(II/I) redox couple, we have constructed mutants of azurin in which the type 1 copper center has been converted to a type 2 center through removal of the equatorial cysteine ligand. These proteins have been further altered through substitutions to the axial methionine at position 121. Through SAM-mediated cyclic and square wave voltammetry, we have demonstrated that the type 2 copper reduction potential in azurin is also tuned via residue 121. In studying these azurin C112D/M121X proteins, we made the observation that in the case of M121L, the axial hyperfine signal in the X-band EPR is significantly narrowed relative to the other axial mutant proteins. At 100x10-4 cm-1, this places the metal center at the extreme edge of the type 1 classification. Through voltammetry measurements, we found that in addition to possessing a reduction potential of 282 mV versus NHE, the rate of electron transfer between the electrode and the copper center has increased over an order of magnitude relative to azurin C112D. These findings suggest that despite the absence of sulfur ligation, copper proteins may adopt type 1 character. We propose to use EXAFS to examine the changes that occur in the oxidized and the reduced forms of C112D/M121X azurins relative to C112D azurin. Precise metal-ligand bond distances for the Cu(I) and Cu(II) proteins will provide insight into the contributions of covalency and reorganization energy.